Project Summary/Abstract A diverse array of chemicals including certain chemical warfare agents (e.g., sulfur mustard), certain intravenous chemotherapy drugs (e.g., mechlorethamine, MEC; paclitaxel, liposomal doxorubicin, ifosfamide, bendamustine), and certain environmental toxins (e.g., viper snake venom metalloproteinases, blister beetle cantharidin) cause acute and severe cutaneous toxicity including dermal-epidermal junction (DEJ) disruption and blistering (vesication). The cutaneous mechanisms involved in chemically-induced DEJ disruption by these types of disruptors remain obscure and current treatments aimed at reducing DEJ disruption remain grossly inadequate. The primary aim of the present proposal, Specific Aim 1, is to investigate novel mechanisms involved in cutaneous responses to DEJ disruptors. Recently, dysregulation of the mammalian target of rapamycin complex 1 (mTORC1) has been implicated in cutaneous injury; with the observation that mouse skin deficient in epidermal expression of Raptor, a component of mTORC1, exhibits epidermal detachments consistent with vesication. Based on this important information, here we will test the hypothesis that DEJ disruption by MEC, a prototype DEJ disruptor, involves both time- and dose- dependent decreases in the epidermal expression of Raptor and phosphorylated forms of S6K and 4E-BP-1. To this end, we will utilize two relevant and complementary skin models: (a) the mouse ear vesicant model (MEVM; in vivo model) and (b) a human skin full thickness 3D culture (in vitro model). Increasing doses of the prototype DEJ disruptor MEC will be applied to mouse ear skin or human skin and punch biopsy samples will be evaluated for cutaneous responses at 0.5, 1, 2, 4, 8, 12 and 24 h after exposure. Dermatotoxicity caused by MEC will be investigated both histologically (by microscopic analyses of edema/ hyperplasia/ inflammation/ vesication) and mechanistically (through the use of immunohistochemical analyses of tissue expression of Raptor and phosphorylated forms of S6K and 4E-BP1 in the epidermis). This highly innovative approach will allow us to decipher the relationship between tissue blistering by DEJ disruptors and components of the mTORC1 signaling pathway. In addition to Specific Aim 1, there are two sub-aims associated with this proposal (Sub-Aim 1 and Sub-Aim 2, respectively). In Sub- Aim 1, the cutaneous expression of several key inflammatory markers (TNF?, IL-1?, iNOS, IL-6, MMP-9) and mIR-132, a microRNA abundant in skin and involved in epidermal cell proliferation and wound healing, will be evaluated in all tissue samples (mouse and human) using qPCR and immunohistochemistry (IHC). In Sub-Aim 2, the time- and dose-dependent effects of MEC on DNA damage sensing and repair responses of the skin will be investigated. DNA damage by MEC will be assessed in all tissue samples (mouse and human) by using immunofluorescence for the presence of DNA damage foci containing phosphorylated ?H2AX (the minor H2 histone variant X), and the repair enzymes FEN1 (flap endonuclease 1) and APE1 (apurinic/apyrimidinic endonuclease 1. Mitochondrial DNA (mtDNA) damage by MEC will be investigated by qPCR. All in all, the work proposed here will establish an important foundation of cutaneous responses to MEC and will aid in deciphering novel targets for future drug development designed to eliminate DEJ disruption by other drugs/chemicals.